Since the initial identification of TNF-.alpha. and TNF-.beta., these proteins have become representative of a unique superfamily of ligands which currently includes TNF-.alpha., TNF-.beta., Fas ligand (L), OX40L, CD40L, CD27L, CD30L, 4-1BBL and LT.beta. (Baker et al. (1996) Oncogene 12:1-9). Each of the ligands is a type II membrane protein which is characterized by the confinement of the C-terminus of the protein within the extracellular space. A 150 amino acid region within the C-terminus is actually the hallmark of the ligand family, as this is the region which the ligands bind to their cognate receptors. In contrast to other protein families with notable homology, each of these family members displays no more than 20-25% homology (which is found mostly in the extracellular region) at the protein level. Although these proteins, for the most part, exist as multimeric membrane bound proteins which function to induce receptor aggregation, there are a few members, such as TNF-.alpha. and FasL, which are functional in a soluble form. Regardless of conformation, these ligands, in a mechanism analogous to other cytokines and growth factors, exert their effects through receptor-ligand interactions which induce downstream signal transduction events.
There are slightly more members of the TNF receptor (TNFR) superfamily as there are known ligands with which they interact: TNF-RI (p55), TNF-RII(p75), TNF-RIII (TNF-RP), OX-40, 4-1BB, CD40, CD30, CD27, the poxvirus gene products PV-T2 and PV-A53R, and p75 BGFR. The mammalian TNFR family members are type I membrane proteins and, in spite of their low degree of homology (20-25%), are grouped together as such due to the presence of conserved cysteine residues in the extracellular ligand-binding domain. In general, each receptor contains varying numbers of cysteine-rich domains (CRDs), each of which is characterized by the presence of approximately 6 cysteine residues that are interspersed within a stretch of 40 amino acids. The presence of the CRDs, based on available crystallographic data for TNF-RI, has allowed this protein superfamily to add a different perspective from which these (as opposed to other) growth factor receptors are studied: a functional TNF superfamily is typically a trimeric or multimeric complex which is stabilized via intracysteine disulfide bonds that are formed between the CRDs of individual subunit members (Banner et al. (1993) Cell 73:431-445). Despite an emphasis on the CRDs and the formation of membrane bound aggregated complexes, most receptors also exist in a soluble form. Solubility is, for the most part, achieved by proteolytic cleavage and soluble forms of TNF-RI, TNF-RII, CD27, CD30, CD40 and Fas are generated in this fashion; while 4-1BB also exists in a soluble form, it is generated via alternate splicing (Gruss et al. (1995) Blood 85:3378-3404).
Irrespective of ligand and receptor conformation, both TNF-related ligands and receptors are expressed on (but necessarily limited to) activated T cells and are, in one form or another, required during T cell mediated immune responses. This type of coordinated expression and function is thought to ensure that such responses, which are largely dependent upon antigen stimulation and subsequent cell-cell interactions, are initiated at the proper times, in the appropriate places, and involve the correct cell types (each of which will express either the ligand or the receptor). Frequently the outcome of such cellular responses can be quite pleiotropic, generating a broad range of cellular responses in the forms of T-cell activation and death, cellular proliferation and differentiation, or cell death which proceeds via apoptosis (Armitage (1994) Cur Opin Immunol 6:406-413; Gruss et al., supra).
Sequence and functional analysis of the various TNF receptor members has revealed a conserved region occurring in the cytoplasmic tail of the Fas and TNF-RI receptors. The term "death domain" was originally coined in 1993 by Tartaglia et al. (Tartaglia L. A. et al., (1993) Cell 74:845-853) as a result of deletion mutagenesis studies involving TNF-RI (p55) mediated apoptotic cell death. These studies revealed that an 80 amino acid domain, which is localized to the C-terminal portion of the protein's intracellular region, is responsible for the generation of cytotoxic death signals, anti-viral responses (Tartaglia et al., supra), and the activation of acid sphingomyelinase (Wiegmann K. et al., (1994) Cell 78:1005-1015); it is also partially responsible for, in conjunction with residues in the N-terminal portion in the intracellular region, the induction of nitric oxide (NO) synthase activity (Tartaglia et al., supra). Homology searches have revealed that the TNF-RI death domain is approximately 65% similar (28% identical) to a 65 amino acid region within the intracellular domain of the Fas antigen; mutagenesis studies have confirmed that these 65 amino acids are required for the induction of cell death following treatment with an anti-Fas antibody in conjunction with actinomycin D (Itoh et al. (1993) J. Biol. Chem. 268:10932-10937). Supporting evidence for a functional overlap between the domains of these two receptors was achieved through the generation of a "death signal delivering" chimeric receptor which replaced TNF-RI amino acid residues 324-326 with the corresponding amino acids of the Fas antigen (Tartaglia et al., supra).
The death domain, aside from being the only homologous intracellular domain that is shared by two members of the TNFR superfamily, generates a cytotoxic signal irrespective of its position with respect to the extracellular domain (Tartaglia et al., supra). In addition, this domain appears to mediate self-association of both TNF-RI and Fas, thereby mimicking the aggregation of events which are induced by ligand binding to each of these receptors (Boldin M. P. et al., (1995) J. Biol. Chem. 270:387-391). These results, which demonstrate that the death domain is an independent domain at both the structural and functional levels, were recently confirmed by the identification and subsequent characterization of three death domain-containing proteins, each of which can generate an apoptotic signal when overexpressed in cells.
The rapid induction of cell death via the death domain is heretofore unique to TNF-RI and Fas; however, despite the characterization of a defined "death inducing" region within each of these receptors, the intermediates involved in the transmission of their signals were, until recently, completely unknown. As with other receptors which are devoid of catalytic activity, TNF-RI and Fas were suspected to utilize cellular protein as "downstream messengers of death." Many charged residues that are well conserved in both proteins were suspected to be widely dispersed throughout portions of the death domains which are oriented to interact with protein components of the cytoplasm (Tartaglia et al., supra). To date, three death domain-containing proteins which associate with either TNF-RI or Fas have been identified and characterized with respect to their ability to induce apoptosis and other downstream signaling events which are activated in immune responses achieved through ligand binding to each of these receptors.
The biologic role of the death domain of TNF-RI and Fas in apoptosis suggests that they may function as tumor supressors. For example, inactivation of Fas signaling as a consequence of loss of Fas/Fas ligand expression/function may lead to abnormal cellular survival and contribute to the development or progression of malignancy as a result of the failure to undergo apoptosis.